A conventional biosensor consists two major components, molecular recognition and signal transduction unit. While specificity is achieved through analyte recognition, the sensitivity is determined by the signal transducer that converts recognition events to measurable physiochemical signals1-3. In existing biosensors, analyte recognition and signal amplification are often decoupled spatiotemporally, such as ELISA (Enzyme Linked Immuno Sorbent Assay)4 these two units require different sets of infrastructure. Although such a design avoids crosstalk between two basic components in a biosensor, the physical separation deteriorates temporal response of the sensors. Moreover, additional steps considerably increase complexity in the traditional sensing, which leads to additional errors that propagate through multiple stages and generate false positive or false negative results.
To address this issue, we have pioneered single-molecule mechanochemical sensing (SMMS)5 in which mechanochemical coupling has been exploited to directly connect molecular recognition and signal transduction units. Mechanochemical coupling is often accompanied by a variation in mechanical properties, such as tension, in the macromolecule upon binding of a ligand. The change in the mechanical property can be monitored in real time using a laser tweezers instrument, accomplishing the sensing without extra infrastructure used in a separate signal transduction unit. Due to synchronizing issues, mechanochemical coupling is most conveniently observed in single-molecule templates. Although such templates offer the utmost sensitivity for individual molecules, which, in theory, can break the detection limit set by the binding affinity between an analyte and a recognition unit employed in ensemble-averaged sensors, the single-molecule platforms have limited space to accommodate expanded functionalities to improve the sensing. For instance, at extremely low analyte concentrations, binding events become so rare that waiting time is beyond experimental reach. To transform single-molecular sensing into highly competitive tools with expanded capabilities, here, we put forward a new concept, single-molecule mechanoanalytical real time sensing device (or SMARTS) that incorporates multiple functional units. We used this SMARTS device to detect Hg2+ ions. Mercury contamination is a prevalent environmental concern as Hg2+ is highly toxic by mutating genomic DNA through binding with a T-T mismatch pair in dsDNA6,7. Currently, Hg2+ is determined by atomic absorption spectrometry (AAS) techniques with a detection limit at sub ppb (1.0×105 fM) levels8,9. Such a level is close to the threshold of 2 ppb considered to be safe by EPA10. However, AAS and other Hg2+ detection methods such as inductively coupled plasma-mass spectrometry (ICP-MS)11, inductively coupled plasma-optical emission spectrometry (ICP-OES)12, and cold vapor-atomic absorption spectrometry (CV-AAS)13 often employ complex procedures that necessitate trained personnel to run the tests14. Recently, Hg2+ has also been detected using electrochemical15,16, optical17, fluorescence18,19, and colorimetric19 detections. To the best of our knowledge, the detection limit of the SMARTS demonstrated here (1 fM in 20 minutes) is at least 2 orders of magnitude lower compared to the most sensitive method described in literature9. The SMARTS has a detection limit 9 orders of magnitude lower than the Kd, which represents at least 3 orders of magnitude improvement in sensitivity with respect to amplification based sensing20 such as ELISA21.